Assessing Toxicity of Waterpipe Tobacco Smoking in Laboratory and Naturalistic Settings Waterpipe (WP) smoking is a significant emerging public health problem in the United States. WP smoking is a tobacco use method in which smoke passes through a partially-filled water jar. Burning charcoal heats the WP tobacco, producing smoke that the user inhales. Exposures to toxicants classified by FDA as harmful and potentially harmful compounds (HPHCs) are 10-100 times higher in WP tobacco smoke in comparison to cigarette smoke. To inform the FDA on the regulation of WP tobacco products, and reduce the harm of this tobacco use method we propose 2 Projects. WP project I. A machine-smoking laboratory study designed to determine the differential effects of emerging WP smoking practices on the toxicity of WP tobacco smoke inhaled by the user (mainstream smoke). Using the Battelle research-grade waterpipe (RWP), and a validated puffing regime, Battelle will machine smoke a popular U.S. manufactured WP tobacco, Starbuzz, to quantify and compare levels of mainstream carbon monoxide (CO), nicotine, select volatile and semi-volatile organic compounds including furans across 4 WP configurations using quick-light charcoal vs. charcoal-free electrically heated WP head, under two conditions for each practice, room temperature water vs. adding ice cubes in the WP jar. WP project II. A naturalistic setting study designed to determine the differential effects of these smoking practices on biomarkers of toxicants and carcinogens related to WP tobacco smoking at home, using a repeated measures design. We will recruit a sample of 50 adult male and female exclusive WP smokers and a control sample of 25 male and female non-smokers, via intercept interviews in San Diego County, CA communities. WP smokers will smoke one WP tobacco head (10g) of Starbuzz during 3 separate sessions with a 7-day washout period before each session using two WP smoking practices: quick-light charcoal vs. charcoal-free electrically heated WP head, under two conditions for when using charcoal, room temperature water vs. adding ice cubes in the WP jar to collect a) Tobacco Use History; b) 4-week Tobacco Exposure Diary; c) Waterpipe Use Session Form; d) CO exposure: Micro+ Smokerlyzer CO monitor will be used for exhaled CO 2 minutes pre and post each smoking session; and e) 6 first morning urine samples: pre and post the 3 sessions to measure urinary cotinine, a metabolite of nicotine, 4-(methylnitrosamino)-1- (3-pyridyl)-1- butanol (NNAL) and NNAL-glucuronides (total NNAL), metabolites of the lung carcinogen 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 1-hydroxypyrene (1-HOP), a metabolite of the genotoxic carcinogen pyrene, and S-phenylmercapturic acid (SPMA), a metabolite of the human hematotoxicant and leukemogen benzene. We will also explore exposure levels to furan, a liver toxicant, among WP smokers via measuring its urinary metabolite Furan-BDA-NAL. Overall analyses include quantifying and comparing mainstream tobacco smoke HPHCs across emerging WP smoking practices; and comparing biomarkers of toxicity of WP smoking across these WP tobacco smoking practices. Key words: Waterpipe, VOCs, Cotinine, NNAL, SPMA, 1-HOP